The peptide hormone insulin stimulates the uptake and storage of glucose and other nutrients into adipose tissue and skeletal muscle while simultaneously repressing glucose efflux from the liver. Insulin resistance occurs when a normal dose of the hormone is incapable of eliciting these anabolic responses, and the condition is a risk factor for cardiovascular disease, type 2 diabetes, and certain forms of cancer. The accumulation of ectopic fat in skeletal muscle has been strongly implicated in insulin resistance, but the mechanism by which excess lipid alters insulin sensitivity is not clear. In particularly, though intramyocellular levels of certain lipid metabolites (e.g., triacylglcyerol and diacylglycerol) correlate with insulin resistance, these molecules don't appear to be active antagonists of insulin action. A long-term goal of our laboratory is to identify the key lipid metabolites which alter insulin sensitivity, and to elucidate the molecular mechanisms by which they inhibit insulin action and disrupt glucose homeostasis. Using experimental systems to deliver exogenous fats to muscle either in vitro or ex vivo, we have determined that the metabolic processing of fatty acids is a key determinant for how they antagonize insulin action. For example, the saturated fatty acid palmitate is almost exclusively reliant on ceramide for its induction of insulin resistance. By contrast, the unsaturated fatty acid linoleate is ceramide-independent, and seems to rely on a glycerolipid intermediate. In the studies proposed herein, we will (a) identify the lipid intermediates which mediate palmitate and linoleate-induced insulin resistance, (b) elucidate the intracellular sensors which link these metabolites to the antagonism of insulin action, and (c) determine how the regulation of fatty acid metabolism by inflammatory factors alters the production of these key lipid metabolites. To obtain this information, we will test the following hypotheses. Aim One: Ceramide, and not a glucosylated ceramide metabolite, links saturated fatty acids to the induction of insulin resistance through the target protein I2PP2A. Aim Two: Phosphatidic acid, and not diacyglycerol, links unsaturated fatty acids to the induction of insulin resistance through the target protein mTOR. Aim Three: Toll like receptors, which are direct effectors of saturated fats, influence insulin sensitivity by diverting incoming fatty acids towards ceramide synthesis. Collectively, these studies could will provide an enhanced understanding of how lipid dysregulation underlies insulin resistance, and could give rise to therapeutic strategies for enhancing insulin sensitivity and combating diabetes. PUBLIC HEALTH RELEVANCE: Excessive deposition of fat in tissues that are not particularly well-suited for fat storage likely contributes to a number of the pathogenic consequences of obesity. However, it is not clear which types of lipid metabolites are deleterious, nor is it understood how these fat derivatives induce cell dysfunction. Using various strategies to alter metabolic pathways controlling fat utilization, we will investigate the role of specific fats in the onset of diabetes.